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Objective:To investigation of the effect on Toll-like receptor-4 (TLR4) mediated signal transduction and the associated factor in periphery blood monocytes due to the activation ofα-adrenoceptor, to explore the interaction ofα-adrenoceptor and TLR4 and the regulatory mechanism involved.Methods:Blood samples were collected from healthy young people at periphery and freeze-conserved for in vitro experiments. (1) To examine the effect ofα-adrenoceptor agonist norepinephrine on protein and mRNA of TLR4, P38mRNA, NF-κBp65 in monocytes of periphery blood and inflammation factor interleukin-6 (IL-6) and interleukin-18 (IL-18) in serum. The blood donors were 10 healthy young people and collected 32 ml periphery venous blood individual. After treatment with anticoagulant EDTA, The blood samples were grouped to blank group, 25μmol/L NE group, 50μmol/L NE group and 75μmol/L NE group. And after incubation with different dose of NE above, ELISA method and flow cytometry was used to determine the concentration of IL-6 and IL-18 in serum and the percentage of TLR4 positive CD14+ monocytes, irrespectively. Determination the expression of TLR4mRNA, P38mRNA use RT-PCR and the expression of NF-κBp65 use immunocyto-chemistry method. (2) After being TLR4mRNA silencing mediated by siRNA method, observation the concentration influence of associated factors stimulated byα-adrenoceptor agonist norepinephrine. The blood donors were 10 healthy young people and collected 16 ml periphery venous blood individual. After treatment with anticoagulant EDTA, The blood samples were grouped to blank group, 75μmol/L NE group, 1 nmol Scramble RNA + 0.24ml transfection system group and 1nmol TLR4 siRNA+0.24ml transfection system group. Determination the concentrations of IL-6 and IL-18 in serum, and the expression of NF-κBp65 in monocytes using ELISA method andimmunocyto-chemistry method, after being transfected for 18 h. (3) After blocking theα-adrenoceptor, P38MAPK, PKA, PKC using respective, simulate withα-adrenoceptor agonist NE, and then observe the expression of TLR4. The blood donors were 10 healthy young people and collected 6 ml periphery venous blood individual. After treatment with anticoagulant EDTA, The blood samples were grouped to 25μmol/Lα-adrenoceptor blocker phentolamine group, 15μmol/L P38MAPK inhibitor SB 202190 group, 15μmol/L pKA inhibitor H89 group, 15μmol/L pKC inhibitor Go6983 group and blank group, 75μmol/L NE group. After incubation for 0.5 h, then add 75μmol/L NE into incubation system, and determine the percent of positive CD14+ monocytes by using flow cytometry.Results:1. After human periphery blood monocytes treated withα-adrenoceptor agonist NE respestively, the expression of TLR4 and mRNA, P38mRNA, NF-κBp65 in nucleus, IL-6 and IL-18 in serum were increased and dose-dependent. And the result showed that the expression value of NE75, NE50 group were distinct increased when compared with the expression value of corresponding blank group (p<0.05,p<0.01). Besides, the expression value of NE75 group was distinct increased than NE75 group (p<0.05), and the expression value of NE25 group and blank group showed no significant difference (p>0.05). 2. After the blood samples treated with noradrenaline, TLR4 Scramble mRNA or TLR4SimRNA described in the Methods, the expression of NF-κBp65, IL6 and IL18 in serum of si+NE75 group were reduced when compared with the expression of blank group, Sc+ NE75 group and NE75 group (P<0.05). 3. After the blood samples treated with noradrenaline, phentolamine, esmolol, H-89, go-6983 or SB20110 to blockα-adrenoceptor, then stimulate with NE (75μmol/L) and detect the expression of TLR4. The results showed that the expression value ofα-blocker group, P38 group, PKA group were reduced when compared with NE75 group (p<0.05), and showed no significant difference with blank group (p>0.05). Besides, the expression value of PKC group, NE75 group were increased compared with blank group (p<0.05).Conclusion:1. Theα-adrenoceptor agonist could induce the activation of TLR4 signaling pathway and revealed a dose-dependent manner, 75μmol/L of norepinephrine was the best dose.2. Theα-adrenergic receptor, P38MAPK、PKA and NF-κB P65 mediated the activation of TLR4 signaling pathway induced byα-adrenoceptor agonist.
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